Means, method and computer program product for determining the concentration level of microorganisms during a fluid analysis

ABSTRACT

A locating method and system of determining a microorganism concentration of a fluid in a Petri dish is disclosed. The Petri dish includes a culture medium on which a seeding device is rotatable relative to the Petri dish. The seeding device distributes the fluid and includes at least one point of contact with the culture medium. The point of contact is associated with a contact zone.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national stage filing under 35 U.S.C. § 371of International application No. PCT/EP2014/050999 filed on Jan. 20,2014 that claims the benefit of French Application No. 1350503 filed onJan. 21, 2013, the entire contents of which are incorporated herein byreference in their entirety.

TECHNICAL FIELD

The present invention relates to the field of biological analysis and,more precisely, a means, a method and a computer software package fordetermining the concentration level of microorganisms present in afluid, during the analysis of a sample of this fluid.

STATE OF THE ART

In the field of biological analysis, there exist numerous methods fordetermining the concentration levels of microorganisms in biologicalsamples. These methods are notably applied by using a Petri dish and aseeding device, such as a suitable comb as described in patentapplications WO2005/071055 and WO2008/093439.

Thus, in the prior art, the Petri dish contains a culture medium such asan agar medium. A fluid sample comprising microorganisms is introducedonto the culture medium. The fluid is applied in a strip which extendsfrom the centre of the Petri dish to the edge of the Petri dish. Thelength of this strip of fluid therefore extends in the direction of theradius of the Petri dish. Then, in the case of a manual or automaticseeding, a comb, such as described in patent applications WO2005/071055and WO2008/093439, moves the microorganisms amassed within the strip offluid, onto the entire circumference of the Petri dish. The comb, whichis situated above the Petri dish, can move in an axis perpendicular tothe plane of the Petri dish, said Petri dish being able to rotate on itsown axis. Generally, the width of the comb corresponds approximately tothe radius of the Petri dish. The comb is positioned such that a firsttooth of the comb is situated at the centre of the Petri dish and a lasttooth of the comb is situated close to the edge of the Petri dish. Thusthe comb makes it possible to distribute the contents of the strip offluid at an angle from 0° to approximately 330° within the culturemedium.

In the case of a manual or automatic seeding, several methods ofdetermining the microorganism concentration are applicable. One of thesemethods employs a division of the Petri dish into eight sectors, i.e.into octants, on a ChromID™ CPS® medium (Ref. 43541) for theidentification and the counting of urinary germs. Thus, when the userwishes to evaluate the bacterial concentration level of the fluiddistributed on the culture medium, the user estimates the culture mediumcover rate in each octant, then uses a chart which has been previouslydrawn up and supplied to the user. This method is laborious because theuser must survey all the colonies present in all of the octants. Thissurveying can therefore generate counting errors, and consequentlyerrors in terms of the bacterial concentration level to be determined.

As the comb moves, the distribution of the fluid on the culture mediumis not homogenous within a given sector, depending on the comb toothunder consideration. Indeed, for an angle of rotation associated withthe movement of the comb, the comb teeth travel a distance which variesdepending on the location of the teeth on the comb. If we consider theteeth situated close to the centre of the Petri dish, the azimuthaldistance travelled by these teeth is less than the azimuthal distancetravelled by teeth situated close to the edge of the Petri dish, for anidentical angle of rotation of the comb on the culture medium.

Thus, applying the method which employs the division of the Petri dishinto eight sectors, the distance travelled by the fluid for a sectorunder consideration depends on the comb tooth associated with themovement of said fluid. Consequently, each sector contains a quantity offluid of which the bacteria concentration is variable on the surface ofa given sector.

The non-homogenous distribution of the fluid within different sectorsalso generates measurement errors when determining the microorganismconcentration present in the fluid to be analysed. Thus, this methoddoes not make it possible to precisely determine the microorganismconcentration within the fluid.

STATEMENT OF THE INVENTION

The present invention is aimed at overcoming the above-mentionedproblems at least in part.

An objective of the present invention consists in supplying a locatingmeans for determining the microorganism concentration of a fluid to beanalysed by means of a Petri dish, said Petri dish comprising a culturemedium on which a seeding device able to rotate relative to the Petridish is fitted so as to distribute the fluid, said seeding devicecomprising at least one point of contact associated with a contact zonewith said culture medium, said locating means comprising:

-   -   a circle C1 corresponding to the circumference of the Petri        dish, the circle C1 comprising a centre O and a radius RC1        corresponding to the radius of the Petri dish;    -   a seeding line LR of length R representing the zone for        inoculating the fluid on the culture medium and corresponding to        a portion of the radius RC1 of the Petri dish;    -   at least one locating zone Zn for locating the presence of        microorganisms on the culture medium,    -   said locating zone Zn being delimited by the seeding line LR and        a zone boundary line LZ delimiting an identical distance d        travelled on the surface of the culture medium by each point of        contact of the contact zone of the seeding device starting from        the seeding line LR.

Advantageously, the fluid comprises a microorganism concentrationgreater than 10³ microorganisms per milliliter (ml).

Advantageously, each locating zone Zn corresponds to a predeterminedvalue of the microorganism concentration of the fluid.

Advantageously, the predetermined value is defined by an exponentialfunction of the angle of rotation of the seeding device, of the length Rof the seeding line and of a constant defining the depletion rate of thebacterial concentration in the fluid as a function of the distancetravelled on the culture medium.

Advantageously, the locating zone Zn is delimited by the circle C1 andby a circle of radius RC2 which is concentric to circle C1 and such thatRC2 is smaller than RC1.

Advantageously, the zone boundary line LZ is formed by a set of pointsPn, each point Pn being situated on a circle Cn concentric to circle C1,said circle Cn comprising a radius RCn, such that RCn is smaller thanRC1 and greater than RC2, and each point Pn being situated at an equaldistance from the seeding line LR for a same zone boundary line LZ.

Advantageously, the microorganisms comprise bacteria.

Advantageously, the culture medium comprises an agar medium.

Advantageously, the fluid consists of a sample of clinical, food,environmental, veterinary, pharmaceutical or cosmetic origin.

Another objective of the invention consists in supplying a locatingmethod for determining the microorganism concentration of a fluid to beanalysed by means of a Petri dish with radius RC1, said Petri dishcomprising a culture medium, said method comprising the following steps:

-   -   depositing the fluid on the culture medium to form a seeding        line LR corresponding to a portion of the radius RC1 of the        Petri dish;    -   distributing the fluid contained in the seeding line LR onto the        culture medium by means of a seeding device able to rotate        relative to the Petri dish, said seeding device comprising at        least one point of contact with the culture medium;    -   incubating the Petri dish to permit the growth of the        microorganisms on the culture medium;    -   identifying adjacent locating zones Zn to locate the presence of        microorganisms on the culture medium, said locating zones Zn        covering at least part of the surface of the Petri dish,    -   and in which a first locating zone Zn is delimited by the        seeding line LR and a zone boundary line LZ delimiting a same        distance d travelled on the culture medium, by each point of        contact of the seeding device starting from the seeding line LR.

Advantageously, the fluid comprises a microorganism concentrationgreater than 10³ microorganisms per milliliter (ml).

Advantageously, the locating method comprises the following step:

-   -   identifying a locating zone Zn comprising isolated colonies of        microorganisms.

Advantageously, the locating method comprises the following step:

-   -   finding, in a correspondence table, a predetermined value for        microorganism concentration associated with the identified        locating zone Zn in order to determine the microorganism        concentration in the fluid.

Another objective of the invention consists in supplying a computersoftware package comprising software instructions for implementing anabove-mentioned locating method when said program is executed by a dataprocessor.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention, its functionality, its applications and its advantagesshall be better understood upon reading the present description, madewith reference to the figures, wherein:

FIG. 1 shows a schematic representation of a locating means, accordingto an embodiment of the invention, by way of example;

FIG. 2 shows an initial detailed schematic representation of a locatingmeans under development, according to an embodiment of the invention, byway of example;

FIG. 3 shows a second detailed schematic representation of a locatingmeans under development, according to an embodiment of the invention, byway of example;

FIG. 4 shows a schematic representation of a locating means integratedonto the lid of a Petri dish;

FIG. 5 shows a Petri dish base comprising an agar medium, according toan embodiment of the invention, by way of example;

FIG. 6 shows a seeding device, such as a comb, situated above a Petridish base, according to an embodiment of the invention, by way ofexample;

FIG. 7 shows a fluid strip placed on the culture medium on the base ofthe Petri dish, according to an embodiment of the invention, by way ofexample;

FIG. 8 shows a diagram of the steps of the locating method, according toan embodiment of the invention, by way of example;

FIGS. 9, 10 and 11 show examples of application of the locating meansonto the contents of various Petri dishes containing various types offluids with various concentrations of microorganisms;

FIGS. 12, 13 and 14 show examples of application of the locating meansonto the contents of various Petri dishes during the analysis of varioustypes of fluid over time.

DETAILED DESCRIPTION OF THE INVENTION

The detailed description below aims to set out the invention in a mannerwhich is sufficiently clear and complete, notably with the aid ofexamples, but must by no means be regarded as limiting the scope ofprotection to the particular embodiments and to the examples presentedbelow.

The present invention relates to the analysis of a sample. According tothe present invention, the sample may be from various origins, forexample of food, environmental, veterinary, clinical, pharmaceutical orcosmetic origin.

Amongst the samples of food origin, non-exhaustive mention can be madeof a sample of dairy products (yogurts, cheeses), meat, fish, egg,fruit, vegetable, water, beverages (milk, fruit juice, soft drink,etc.). Of course, these samples of food origin can also come from saucesor more complex dishes, or from processed or partially processed rawmaterials. A food sample can also come from an animal feed, such as oilmeals or animal meals.

As indicated previously, the sample can be of environmental origin andcan consist, for example, of a surface sample, water sample, air sample,etc.

The sample can also consist of a sample of clinical origin, which cancorrespond to specimens of biological fluid (urine, whole blood orderivatives such as serum, saliva, pus, cerebrospinal fluid, etc.), ofstools (for example cholera-induced diarrhoea), of specimens from thenose, throat, skin, wounds, organs, tissues or isolated cells. This listis obviously not exhaustive.

Generally, the term “sample” refers to a part or a quantity, and moreparticularly a small part or a small quantity, taken from one or moreentities for the purpose of analysis. This sample can possibly haveundergone pretreatment, involving for example steps of mixing, dilutingor even crushing, in particular if the starting entity is in the solidstate.

The sample collected is, in general, capable of—or suspectedof—containing at least one target external microorganism such as abacterium, a yeast or a fungus.

According to the present invention, the analysis is carried out using aPetri dish comprising a base and a lid. The base comprises a culturemedium such as an agar culture medium. The aim of the analysis is todetermine the presence of specific microorganisms and the concentrationof microorganisms within the fluid on the culture medium, afterincubation of the Petri dish.

The present invention proposes determining the concentration of themicroorganisms present on the culture medium by locating the colonies ofmicroorganisms isolated on said culture medium.

FIG. 1 shows a locating means 10. This locating means 10 can be madefrom a translucent material in order to be placed on the lid of a Petridish to observe the agar culture medium containing the microorganisms.The locating means 10 can also be integrated into the lid of a Petridish by means of a graphic method, for example. Finally, the locatingmeans 10 can also be integrated within a computer software in order toenable analysis of the microorganism presence by means of a computer.

The locating means 10 comprises a circle C1, the dimensions of which areof the same order of magnitude as the dimensions of a circlerepresenting the base of a Petri dish. The circle C1 comprises a centreO and a radius RC1.

The locating means 10 also comprises a circle C2 which is concentric tocircle C1 and the radius RC2 such that radius RC2 is smaller than radiusRC1.

Circle C1 also comprises a seeding line LR of length R extending in thedirection of radius RC1, and such that the length R is smaller than orequal to the value of the difference between radii RC1 and RC2, i.e.R<RC1−RC2. The seeding line LR represents a reference line along which astrip of fluid to be analysed is inoculated on the culture medium.

A seeding device makes it possible to move the fluid from the seedingline LR, onto all or a part of the surface of the agar culture medium onthe base of the Petri dish. The length R of the seeding line LR isgenerally smaller than or equal to the width of the seeding device. Theseeding device comprises a contact zone with the agar culture medium.The contact zone comprises at least one point of contact with the agarculture medium via the fluid situated between the point of contact andthe culture medium. The contact zone may be, for example, continuous ordiscontinuous.

The seeding device comprises for example a comb such as described inpatent applications WO2005/071055 and WO2008/093439. Each tooth of thecomb corresponds to one point of contact. Thus the comb teeth as a grouprepresent a discontinuous contact zone with the culture medium.

The seeding device can also comprise a spatula with curved end such asdescribed in the model U.S. D582,564. Thus, the curved end represents acontinuous contact zone of the seeding device with the agar culturemedium.

During the distribution of the fluid on the culture medium by means ofthe seeding device, each point of contact of the contact zone of theseeding device travels an azimuthal distance starting from the seedingline LR. This azimuthal distance varies according to the point ofcontact under consideration of the contact zone of the seeding device.

The locating means 10 also comprises a set of locating zones Zn whichcover all or a part of the surface of the agar culture medium within thesurface delimited by the circles of radii RC1 and RC2. These zones arenumbered from Z1 to Z7, for example. Each locating zone Zn comprises azone boundary line LZ, which makes it possible to separate two adjacentlocating zones. The disc associated with circle C2 represents a surfacewhich is non-useable for the seeding device. Indeed, for radius valuessmaller than the value of RC2, in the disc of radius RC2, the azimuthaldistances travelled by the points of contact of the contact zone of theseeding device are too small to make it possible to determine themicroorganism concentration using the locating means according to thepresent invention.

The zone boundary lines LZ are labelled LZ1 to LZ7, for example. Theselines are made on the locating means 10 using a method illustrated inFIG. 2.

FIG. 2 shows the locating means 10 during its development to obtain therepresentation of the zone boundary lines LZ.

The locating means 10 comprises circles C1 and C2 as previouslyindicated.

In order to develop the locating means 10, concentric circles Cn ofradius RCn and centre O are traced onto the locating means 10. Thecircles Cn are such that each radius RCn is smaller than radius RC1 andgreater than radius RC2. The presence of the graphic representation ofthe circles Cn is not necessary during the subsequent use of thelocating means 10.

The circumference of circle C1 is divided into a determined number ofcircle portions, where each circle portion represents an azimuthaldistance d. The azimuthal distance d corresponds to a movement of thepoints of contact of the contact zone of the seeding device from theseeding line LR, during a rotation of the seeding device. Ai is definedas being the real or effective angle of rotation of the seeding deviceon circle C1 relative to the seeding line LR. Each azimuthal distance don circle C1 delimits the locating zones as Z1 to Z7. Thus, eachlocating zone Zn is delimited, on the circumference of circle C1, by anazimuthal distance which is a multiple of the azimuthal distance d andpreferably equal to the azimuthal distance d.

Thus, in a known manner, the portion d represents an azimuthal distanceequal to the product of distance R, lower than or equal to thedifference RC1−RC2 and representing the length of the seeding line LR,by the angle of rotation Ai which is predetermined and associated withthe rotation of the seeding device during the movement of the seedingdevice on the circumference of circle C1. Thus, we obtain d=R×Ai.

One of the aims of the present invention is to supply a locating means10 which makes it possible to locate the zones of the culture medium inwhich isolated colonies of microorganisms are observed. Thus, thelocating means 10 makes it possible to determine the initialconcentration of microorganisms in the fluid of interest, i.e. thecarrier liquid containing the bacteria.

For each circle Cn, an azimuthal distance dn is located. This distancedn is the representation of the azimuthal distance travelled by thepoints of contact of the contact zone of the seeding device on aspecific circle Cn during the rotation of the seeding device by an angleAi.

The azimuthal distance dn is therefore equal to a multiple of theazimuthal distance d, the value of which is defined by a determinedvalue. Thus, by referring to the known value of d, we obtaindn=(R×Ai)×k, where k is a number representing the coefficient relativeto the multiplicity of d relative to dn.

Aj is defined as being the fictitious angle of rotation of the seedingdevice on a circle Cn relative to the seeding line LR for a point ofcontact situated at a distance Rn from the centre O, such that theazimuthal distance dn is equal to d for a specific circle Cn.

The azimuthal distance dn is also equal to the product of radius Rn bythe angle of rotation Aj associated with the rotation of a point ofcontact of the contact zone of the seeding device, i.e. dn=Rn×Aj. Thevalue of dn corresponds to the distance travelled by a point of contactof the contact zone of the seeding device on the concentric circle Cnduring the rotation of the seeding device by an angle Aj. Each portiondn makes it possible to represent the distance or path that each pointof contact of the contact zone must travel on a given circle Cn toobtain an azimuthal distance travelled and relative to the zone Zn underconsideration such that dn is equal to d.

The value of d is therefore determinable insofar as the values of theangle of rotation Ai of the seeding device and of the radius Rassociated with the length of the seeding line are known. By usingAj=dn/Rn, the value of the angle of rotation Aj is obtained. Thus, onthe circle Cn, for a rotation of angle Aj starting from the seeding lineLR, a point Pn is obtained which represents the end of the portion dn onthe corresponding circumference of circle Cn.

Thus, as shown in FIG. 3, a rotation of the seeding device by an angleAi represented by a portion d on circle C1 corresponds to a rotation ofthe comb by an angle Aj to obtain a portion of length d2 identical to don circle C2.

Therefore, we obtain Aj=d2/RC2, where d2=d=Ai×R, which makes it possibleto define the angle Aj associated with distance d2 on circle C2, suchthat Aj=(Ai×R)/RC2.

Thus, by repeating this method on all of the circles Cn, there isobtained a point Pn+1 on circle Cn+1, a point Pn+2 on circle Cn+2, andso on on all of the circles Cn as shown in FIG. 2.

For each rotation of the seeding device, the associated points P1 and P2on circles C1 and C2 are defined.

For a same angle of rotation Ai, all of the points Pn, Pn+1, Pn+2, etc.associated with a same zone Zn, are connected together as shown in FIG.2 to form a line. The end of this line is connected to points P1 and P2,respectively situated on circles C1 and C2, to obtain the representationof a locating zone Zn such as shown in FIG. 2. The locating zone Zncontains a quantity of fluid associated with a movement of the seedingdevice over a same distance d.

Thus, during the rotation by an angle Ai of the seeding device, thepoint of contact of the contact zone travels an azimuthal distance d onthe circumference of circle C1. Zone Z1 makes it possible to locate thequantity of fluid which has travelled the same distance d from theseeding line LR. Each zone Zn is located on the circumference of circleC1 by an azimuthal distance equal to d.

The seeding device makes a set number of rotations of angle Ai vis-à-visthe base of the Petri dish. These rotations of angle Ai make it possibleto spread on the culture medium the entirety of the fluid contained inthe seeding line LR.

Conveniently, the locating means 10 may have an independent support tobe applied onto the lid of a Petri dish after incubation of the Petridish. Alternatively, a lid of a Petri dish may integrate a graphicrepresentation of the locating means 10. Thus, FIG. 4 shows a lid 40 ofa Petri dish integrating a graphic representation of the variouslocating zones. FIG. 5 shows a base 50 of a Petri dish containing anagar culture medium. Thus, the lid 40 may be placed on the base 50 inorder to locate the microorganisms on the agar culture medium.

As shown in FIG. 6, a seeding device such as a comb 60 is placed abovethe base 50 of a Petri dish. The base 50 is represented by a circle C1with a centre O. The base 50 can be mounted on a support capable ofmoving in a circular movement in order to produce a rotation of the base50. The base 50 contains a culture medium such as an agar culturemedium. A seeding line LR of length R is placed on the culture medium.

The comb 60 comprises one teeth support 61 equipped with a plurality ofteeth 62 comprising at least one tooth. Preferably, the comb 60comprises 17 teeth. Each tooth 62 is separated from an adjacent tooth bya same distance. The total width of the comb 60 corresponds to a valuesmaller than the length R of the seeding line. The comb 60 is able torotate relative to the base 50 of the Petri dish.

The locating method according to the invention makes it possible tolocate microorganisms present within a fluid on the culture medium,after incubation of the Petri dish. The fluid comprises, for example, abiological sample as mentioned previously. For the purposes of thepresent example, we shall consider the hypothesis wherein the length Rof the seeding line LR is equal to the radius RC1 of the Petri dish.

The method according to the invention comprises the steps described inFIG. 8. In a step 800, an inoculation device (not shown), controlled forexample by a computer program by means of a computer, deposits aquantity of fluid in the form of a seeding line LR onto the culturemedium as shown in FIG. 7. The seeding line LR extends over the base 50of the Petri dish, in the direction of the radius RC1 of the Petri dish.

In a known manner, in a step 810, the comb 60 is applied onto the fluidsituated on the seeding line LR. The comb 60 and the base 50 of thePetri dish are designed to work together in the following manner: duringa rotation of the base 50, the comb 60 comes into contact with theseeding line LR. Thus, in a step 820, the base of the Petri dish startsto rotate and the comb 60 distributes, on the culture medium, the fluidcontained in the seeding line LR. The comb 60 can be preset to make,relative to the base 50 of the Petri dish, repeated rotations centred onthe centre O of the base 50 of the Petri dish, and of a same angle Ai.

During the movement of the comb, only the tooth 21 situated on theperiphery of the base 50 of the Petri dish moves by an azimuthaldistance d on the circumference of the circle C1 such that d=R×Ai. Theother teeth 62 of the comb 60 travel an azimuthal distance, the lengthof which is lower than d. Thus, for a same comb 60, the fictitious angleof rotation Aj of each tooth 62 situated strictly inside the circle C1must be larger than the actual angle of rotation Ai, so that each tooth62 travels an azimuthal distance identical to d on the correspondingconcentric circle Cn. Indeed, as we consider a tooth 62 nearer to thecentre O of the circle C1, the azimuthal distance travelled by the tooth62 decreases, for a given angle of rotation Ai.

The movement of the comb 60 associated with the movement of the base ofthe Petri dish is repeated until the totality of the contents of thefluid of the seeding line LR is distributed on the agar culture medium.

Then, in a step 830, the base 50 of the Petri dish covered by a lidsuitable for a Petri dish is incubated in an incubator for apredetermined period. The aim of the incubation is to enable the growthof microorganisms potentially contained in the fluid distributed on theculture medium. The incubation period can extend over several hours orover several days, preferably over twenty-four hours. This incubationperiod can be continuous, i.e. without intermediate analysis of thecontents of the Petri dish, between the inoculation of the biologicalsample and the end of the incubation period. The incubation period canalso be discontinuous, i.e. with a regular analysis of the contents ofthe Petri dish at predetermined times during the incubation period.

The steps 810, 820 and 830 previously described can be carried out bymeans of an automated device controlled by a computer program by meansof a computer.

At a predetermined time, either during the incubation period or at theend of the incubation period, in a step 840, the locating means 10according to the present invention is applied onto the Petri dish. Step840 can be performed either manually, or automatically by means of amechanical device controlled by a computer program, or electronicallyusing the image of a locating means to be applied onto an image of thePetri dish by means of an electronic device such as a computer.

After incubation, the culture medium, in the base 50 of the Petri dish,contains colonies of microorganisms situated at different locations onthe surface of the culture medium. Certain colonies can appear isolatedand therefore be easily counted visually by the user. Other colonies canbe amassed and be impossible to count. In a situation in which the valueof the microorganism concentration per milliliter (ml) is greater than aconcentration of around 10³ microorganisms per ml, preferably greaterthan a concentration equal to 10³ microorganisms per ml, thedistribution of the colonies is represented by continuous zones ofcolonies on the culture medium. In this case, the use of the locatingmeans 10 according to the present invention is optimal.

In a step 850, the user can visually count the isolated colonies withinthe locating zones of the locating means 10. Step 850 can also beperformed by means of an automated device controlled by a computerprogram with reference to an image processing method, using the image ofa locating means applied onto an image of the Petri dish, by means of anelectronic device such as a computer.

Then, in a step 860, the user searches for the value of theconcentration associated with the number of isolated colonies counted ina zone under consideration using, for example, the correspondence tablebelow:

Concentration in CFU/ml (CFU: Colony Number of isolated colonies locatedin a locating Forming Unit) zone 10² Less than 10 colonies 10³ Less than30 colonies 10⁴ Greater than 30 colonies and locating zone Z1 is notfull 10⁵ Locating zone Z1 filled (no isolated colony) Locating zone Z2and Z3 containing isolated colonies 10⁶ Locating zone Z1 and Z2 filled(no isolated colony) Locating zones Z3 and Z4 containing isolatedcolonies 10⁷ Locating zone Z1, Z2 and Z3 filled (no isolated colony)Locating zone Z4 containing isolated colonies 10⁸ Locating zone Z1, Z2,Z3 and Z4 filled (no isolated colony) Locating zone Z5 containingisolated colonies

The concentration values presented in the table above are obtained usingthe mathematical formula C (RCn, A)=Coexp(−RCn×A/K) which defines thedistribution of microorganism concentration on the culture medium. Inthis mathematical formula:

A is the value of the actual angle of rotation of the seeding device(60);

Co is the initial microorganism concentration of the inoculated fluid;

RCn is the radius of the concentric circle under consideration;

K is a depletion constant specific to a fluid relative to the agarculture medium on which the fluid is distributed.

Step 860 can be performed by means of a computer containing a suitablecomputer program.

Thus, steps 800, 810, 820, 830, 840, 850 and 860 can be performed in anautomated manner.

FIGS. 9, 10 and 11 show examples of application of the locating methodfor urinary samples.

FIG. 9 shows progressive concentrations from 10² to 10⁴ CFU/ml on TSAagar (Tryptic Soy Agar).

FIG. 10 shows progressive concentrations from 10⁵ to 10⁶ CFU/ml on TSAagar.

FIG. 11 shows progressive concentrations from 10⁷ to 10⁸ CFU/ml on TSAagar.

Similarly, FIGS. 12, 13 and 14 show an example of analysis of a samefluid at different concentrations (10² to 10⁸ CFU/ml) inoculated onChromID™ CPS® medium at different successive moments in time. Thus,taking a number of hours as a basis, it is possible to observe theevolution of the microorganism concentration at a given time H, thensuccessively at a time H+10 hours, H+26 hours, and so on, for example.This involves extemporaneous preparation of bacterial solution. Thus,the reproducibility of the locating method can be estimated.

In total, 122 dishes have been tested with the method according to thepresent invention on ChromID™ CPS® medium, i.e. a medium dedicated tourine counting, for which this method is extremely useful. The thresholdconcentration value was 10⁴ CFU/ml.

As shown in the table below, a sensitivity and a specificity of 100% isobtained relative to the theoretical bacterial concentration.

Estimation with the locating means according to the present invention.Negative Uncertainty Positive Total Theory Negative 40 0 0 40 (<10⁴CFU/ml) Uncertainty 0 18 0 18 (=10⁴ CFU/ml) Positive 0 0 59 59 (>10⁴CFU/ml) Total 40 18 59 117

The present invention is specifically suitable for the seeding devicesdescribed previously or for seeding devices which have a similar mode ofoperation concerning the distribution of the fluid on a culture mediumsuitable for the mode of distribution.

Such seeding devices comprise, for example, devices which are able torotate relative to the centre of the Petri dish and which make itpossible to radially deposit the fluid by means of radial extension.

The invention claimed is:
 1. A locating system for determining amicroorganism concentration of a fluid in a Petri dish comprising a lidand a base and a culture medium on which a seeding device is rotatablerelative to the Petri dish to distribute the fluid, wherein the seedingdevice comprises at least one point of contact, associated with acontact zone, with the culture medium, the locating system beingsuitable to be placed on the lid of the Petri dish or integrated in thelid of the Petri dish or integrated within a computer software andcomprising: a first circle corresponding to the circumference of thePetri dish, the first circle comprising a centre and a first radiuscorresponding to the radius of the Petri dish; a seeding line having alength representing the zone for inoculating the fluid on the culturemedium and corresponding to a portion of the first radius of the Petridish; and at least one locating zone for locating the presence ofmicroorganisms on the culture medium, wherein the locating zone isdelimited by the seeding line and a zone boundary line delimiting anidentical azimuthal distance travelled on the surface of the culturemedium by each point of contact of the seeding device starting from theseeding line, and wherein the azimuthal distance corresponds to movementof the at least one point of contact from the seeding line duringrotation of the seeding device.
 2. The locating system of claim 1,wherein the fluid comprises a microorganism concentration greater than10³ microorganisms per milliliter.
 3. The locating system of claim 1,wherein each locating zone corresponds to a predetermined value of themicroorganism concentration of the fluid.
 4. The locating system ofclaim 3, wherein the predetermined value is defined by an exponentialfunction of the angle of rotation of the seeding device, the length ofthe seeding line of the Petri dish, and a constant defining thedepletion rate of the bacterial concentration in the fluid as a functionof the azimuthal distance travelled on the culture medium.
 5. Thelocating system of claim 1, wherein the locating zone is delimited bythe first circle and by a second circle of a second radius which isconcentric to the first circle, and such that the second radius issmaller than the first radius.
 6. The locating system of claim 5,wherein the zone boundary line is formed by a set of points, each pointbeing situated on a third circle concentric to the first circle, thethird circle comprising a third radius, such that the third radius issmaller than the first radius and greater than the second radius, andeach point is situated at an equal distance from the seeding line forthe same zone boundary line.
 7. The locating system of claim 1, whereinthe microorganisms comprise bacteria.
 8. The locating system of claim 1,wherein the delimitation of the locating zone is by the seeding line anda zone boundary line delimiting an identical azumithal distancetravelled on the surface of the culture medium by each point of contactof the seeding device starting from the seeding line.